Nanotubes as mitochondrial uncouplers

ABSTRACT

A method of uncoupling mitochondria in a subject including administering nanotubes to the subject in a therapeutically effective amount, wherein the nanotubes are self-rectifying is provided. A method of decreasing reactive oxygen species and decreasing detrimental loading of Ca 2+  into mitochondria is provided, including administering a pharmaceutically effective amount of nanotubes into the subject. A method of reducing weight, treating cancer, reducing the effects of traumatic brain injury, or reducing the effects of ageing, in a subject including administering a pharmaceutically effective amount of nanotubes into the subject is also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.13/035,649, filed Feb. 25, 2011, which is a divisional of U.S.application Ser. No. 11/418,208, filed May 5, 2006 which claims priorityunder 35 U.S.C. § 119 to U.S. Provisional Application No. 60/678,355,filed May 6, 2005, the entire content of which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to nanotubes as mitochondrial uncouplers.The present invention provides methods of mitochondrial uncoupling aswell as methods of treating disease conditions and increasing weightloss by administering the nanotubes.

BACKGROUND

It is known that mitochondria control metabolism in individual cells byburning sugars and fats. Mitochondria produce a membrane potential ofabout 200 mV across their inner membrane by the active translocation ofprotons from the matrix of the mitochondria (the inside) into the innermembrane space (mitochondria have an inner an outer membrane asillustrated in FIG. 2). The translocation of protons from the matrix isdue to the activity of the electron transport system, which takeselectrons from a high energy state to a lower energy resulting in thereduction of oxygen to water, hence the term mitochondrial respirationsince oxygen is consumed by this process. The energy released aselectrons are taken from a high energy state to a lower energy state isused by this electron transport system to translocate (i.e., pump) theprotons from the matrix to the inner membrane space resulting in aseparation of charge (i.e., membrane potential) as well as a pH gradientacross the inner membrane due to the movement of these protons.

The mitochondrial membrane potential is then “coupled” to the controlledflow of protons back into the matrix through the ATP synthase which usesthis flow to phosphorylate adenosine diphosphate (“ADP”) to ATP.

Chemical uncouplers of mitochondria have been used to increase thebodies basal metabolism to encourage weight loss. However, chemicaluncouplers are readily toxic, as they are difficult to control.Uncontrolled uncoupling of mitochondria (i.e., dropping themitochondrial membrane potential below about 100 mV) causes an inabilityto produce cellular ATP, which eventually leads to death.

Therefore, there is a need for safe, controllable, mitochondrialuncouplers that can separeate the mitochondria respiration from theproduction of ATP, and thus safely produce the desired effect, withoutharming the individual.

SUMMARY OF THE INVENTION

The present invention provides nanotubes which safely uncouplemitochondria, as the nanotubes can be prepared such that the protonchannel automatically shuts down when an unsafe potential is reached.

The template method of forming nanotubes in one embodiment allowscontrol of the dimensions of the nanotubes such that the nanotubes canbe designed to have any desired dimensions as specified. By allowingmanipulation of size, particularly the inner diameter of the nanotubes(as explained above), these nanotubes allow specific conductance (i.e.,uncoupling threshold and resistance to proton flow) of the nanotubes.The template method further allows lining or coating the inside andoutside of the nanotubes with any desired materials.

Template synthesis of nanotubes generally involves the deposition ofmaterials (e.g., gold, alumina, silica etc.) into the cylindrical poresof mass-produced track-etched membranes (e.g., Corning Corp; Osmonics).Once the nanotubes are formed within the membrane pores, the membranecan be dissolved and the nanotubes can be captured via filtration. Byaltering the conditions during preparation (e.g., reducing the platingtime when using the electroless plating method for creating goldnanotubes increases the inner diameter of the nanotube accordingly) theinternal diameter, length and contents of the nanotube can be controlledas specified.

According to another embodiment, nanotubes made of metals and/orpolymers are used to safely uncouple mitochondria, raise metabolism, andpromote weight loss.

According to another embodiment, the nanotubes have a length of about8-12 nm and a diameter of about 1-3 nm, wherein the nanotubes can crosscell membranes substantially easily, and wherein the mitochondria have alength of about 1000-5000 nm and the bilipid membrane of themitochondria is about 8-10 nm thick.

According to a further embodiment, the nanotubes can be active only inthe mitochondria. This is due to the fact that the nanotubes only act asproton channels when a significantly high potential is applied (about130-150 mV), which is only found across the mitochondrial inner membranein mammal cells.

In a further embodiment, the interior surface of the non-carbon basednanotubes can be doped with compounds of specific pKa such that thenon-carbon based nanotube shuts off when a specific pH is reached. Thisallows us to take advantage of the fact that mitochondria have a pHgradient across the inner membrane as a result of the pumping of protonsfrom the matrix across the inner membrane via the electron transportsystem.

In one embodiment, the nanotubes act as proton channels specifically inthe mitochondria. The nanotubes acting as proton channels cause areduction in mitochondrial membrane potentials which in turn increasesbasal metabolism, decreases reactive oxygen species (“ROS”), anddecreases detrimental loading of Ca²⁺ into mitochondria. The reason forthis is that all these parameters are a function of the membranepotential such that a high mitochondrial membrane potential slows downmetabolism and increases ROS formation and the uptake of Ca²⁺ intomitochondria.

The nanotubes conduct protons (i.e., open and uncouple mitochondria)only when a specific potential (i.e., breakover voltage) is reached.This causes a drop in the mitochondrial membrane potential that closesthe nanotube proton channel, in effect making the nanotubesself-rectifying. Therefore, nanotubes can be designed to maintain aspecific mitochondrial membrane potential that can significantlyincrease metabolism without the possibility of toxicity since thenanotubes will not function (act as proton channels) at a mitochondrialmembrane potential below the threshold for ATP production.

According to one embodiment, the dimensions of nanotubes and thematerials they are made from are manipulated to accomplish safemitochondrial uncoupling. For example, when the diameter of thenanotubes is decreased, a smaller diameter increases resistance and theconductance of protons through the nanotubes (uncouples the mitochondriaelectron transport from ATP production) only when the voltage reachesthreshold (the mitochondrial membrane potential). These nanotubes thatare highly effective for weight-loss and at the same time aresubstantially safe for use because they are self-rectifying.

In one embodiment, nanotubes can offer a safe and effective treatmentfor obesity and weight management by increasing basal metabolism byincreasing mitochondrial respiration.

In another embodiment a method of uncoupling mitochondria in a subjectcomprising administering nanotubes to the subject in a therapeuticallyeffective amount, wherein the nanotubes are self-rectifying isdisclosed.

The nanotubes can be coated with a pKa reducing compound. The subjectcan be a mammal. The nanotubes have an inner diameter suitable foruncoupling mitochondria. The nanotubes have an inner diameter whichallows the nanotubes to self-rectify.

The nanotubes comprise metals or polymers, wherein the metal is gold orsilver, and wherein the polymers are natural polymers or syntheticpolymers. The polymers are preferably selected from the group consistingof poly(vinyl alcohol), poly(esters), polyglycolide, polycaprolactone,poly(ethylene oxide), poly(butylene terephthalate),poly(hydroxyalkanoates), hydrogels, modified poly(saccharides), starch,cellulose, chitosan and combinations thereof.

In another embodiment, a method of decreasing reactive oxygen speciesand decreasing detrimental loading of Ca²⁺ into mitochondria of asubject comprising administering nanotubes to the subject in atherapeutically effective amount, wherein the nanotubes areself-rectifying is disclosed.

The subject can be a mammal. The nanotubes have an inner diameterdesigned for uncoupling mitochondria. The nanotubes have an innerdiameter which allows the nanotubes to self-rectify. The nanotubescomprise metals or polymers, wherein the metal is gold or silver, andwherein the polymers are natural polymers or synthetic polymers. Thepolymers are preferably selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.

In another embodiment, a method of reducing weight in a subjectcomprising administering a therapeutically effective amount of nanotubesto the subject, wherein the nanotubes are self-rectifying is disclosed.

The subject can be a mammal. The nanotubes have an inner diameterdesigned for uncoupling mitochondria. The nanotubes have an innerdiameter which allows the nanotubes to self-rectify. The nanotubescomprise metals or polymers, wherein the metal is gold or silver, andwherein the polymers are natural polymers or synthetic polymers. Thepolymers are preferably selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.

In another embodiment, a method of treating cancer in an subjectcomprising administering a therapeutically effective amount of nanotubesto the subject, wherein the nanotubes are self-rectifying is disclosed.

The subject can be a mammal. The nanotubes have an inner diameterdesigned for uncoupling mitochondria. The nanotubes have an innerdiameter which allows the nanotubes to self-rectify. The nanotubescomprise metals or polymers, wherein the metals are gold or silver, andwherein the polymers are natural polymers or synthetic polymers. Thepolymers are preferably selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.

In another embodiment a method of reducing the effects of traumaticbrain injury in an subject comprising administering a therapeuticallyeffective amount of nanotubes to the subject, wherein the nanotubes areself-rectifying is disclosed.

The subject can be a mammal. The nanotubes have an inner diameterdesigned for uncoupling mitochondria. The nanotubes have an innerdiameter which allows the nanotubes to self-rectify. The nanotubescomprise metals or polymers, wherein the metals are gold or silver, andwherein the polymers are natural polymers or synthetic polymers. Thepolymers are preferably selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.

In another embodiment, a method of reducing the effects of ageing in asubject comprising administering a therapeutically effective amount ofnanotubes to the subject, wherein the nanotubes are self-rectifying isdisclosed.

The subject can be a mammal. The nanotubes have an inner diameterdesigned for uncoupling mitochondria. The nanotubes have an innerdiameter which allows the nanotubes to self-rectify. The nanotubescomprise metals or polymers, wherein the metals are gold or silver, andwherein the polymers are natural polymers or synthetic polymers. Thepolymers are preferably selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.

In another embodiment, a pharmaceutical composition is disclosed,wherein the pharmaceutical composition comprises nanotubes in apharmaceutically acceptable carrier, and wherein the pharmaceuticalcomposition is administered to an subject by (i) intravenous delivery,(ii) ingestion, (iii) particle bombardment via a gene gun, or (iv) patchor gel application to the dermis.

In another embodiment a method of reducing the effects spinal cordinjury in a subject comprising administering a therapeutically effectiveamount of nanotubes to the subject, wherein the nanotubes areself-rectifying is disclosed.

In another embodiment, a method of reducing the effects of stroke in asubject comprising administering a therapeutically effective amount ofnanotubes into the subject, wherein the nanotubes are self-rectifying isdisclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two line graphs indicating that 2,4-DNP increasesmetabolism and weight loss in a dose-dependent manner in humans.

FIG. 2 shows bar graphs that illustrate mitochondrial uncoupling as aresult of administering 2,4-DNP increases tissue sparing followingtraumatic brain injury.

FIG. 3 shows bar graphs that illustrate post-injury administration of2,4-DNP increases tissue sparing following spinal cord injury (“SCI”).

FIG. 4 shows electron microscopy photomicrographs of isolated corticalmitochondrial following the addition of nanotubes (24 hour+histidine)that were made in the presence (left panel) or absence (right panel) ofPEG.

FIG. 5 shows a higher magnification of isolated cortical mitochondrialfollowing the addition of nanotubes (24 hour+histidine) that were madein the presence of PEG.

FIG. 6 is a bar graph that shows that mitochondrial respiration isincreased as a function of nanotube plating time (the longer the platingthe time the smaller the diameter of the nanotube) and/or the “doping”of the inside of the nanotube with compounds of specific pKas.

FIG. 7 shows nanotubes manufactured by plating for 24 hrs and/or “doped”with histidine do not impair mitochondrial ATP production.

FIG. 8 shows nanotubes manufactured by plating nanotubes for 24 hrsand/or doping them with histidine increase respirations less thannanotubes manufactured for shorter times.

FIG. 9 shows that sequential additions of nanotubes results in asaturation point being reached due the self-rectifying nature of thenanotubes.

FIG. 10 shows that nanotubes reduce mitochondrial membrane potentialwhich explains their ability to increase respiration, oxygen consumptionand metabolism.

FIG. 11 shows that nanotubes reduce reactive oxygen species productionin isolated mitochondria.

FIG. 12 shows a line graph of days v. grams of weight gained uponadministering nanotubes compared to administering vehicle controls overa period of seven days.

FIG. 13 shows a line graph of days v. grams of weight gained uponadministering nanotubes compared to administering vehicle controls overa period of 31 days.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “compounds” includes a plurality of such compounds andreference to “the dosage” includes reference to one or more dosages andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below:

“Pharmaceutically acceptable carrier” means a carrier that is useful inpreparing a pharmaceutical composition that is generally safe, non toxicand neither biologically nor otherwise undesirable, and includes acarrier that is acceptable for veterinary use as well as humanpharmaceutical use. “A pharmaceutically acceptable carrier” as used inthe specification and claims includes both one, and more than one, suchcarrier.

“Treating” or “treatment” of a disease includes:

-   -   (1) preventing the disease, i.e., causing the clinical symptoms        of the disease not to develop in a mammal that may be exposed to        or predisposed to the disease but does not yet experience or        display symptoms of the disease,    -   (2) inhibiting the disease, i.e., arresting or reducing the        development of the disease or its clinical symptoms, or    -   (3) relieving the disease, i.e., causing regression of the        disease or its clinical symptoms.

A “therapeutically effective amount” or “pharmaceutically effectiveamount” means the amount of a compound that, when administered to amammal, is sufficient to uncouple mitochondria, and/or sufficient todecreasing reactive oxygen species and decreasing detrimental loading ofCa²⁺ into mitochondria. A “therapeutically effective amount” or“pharmaceutically effective amount” also means and amount sufficient fortreating a disease and sufficient to effect such treatment for thedisease. The “therapeutically effective amount” will vary depending onthe compound, the disease and its severity and the age, weight, etc., ofthe mammal to be treated. A “therapeutically effective amount” also mayrefer to an amount sufficient to cause weight loss.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of scopolamine which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance may, but need not, occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not.

The term “individual” is a vertebrate, preferably a mammal. Mammalsinclude, but are not limited to, humans, rodents (i.e., mice, rats, andhamsters), farm animals, sport animals and pets. In a preferredembodiment, the individual is a mammal and more preferably, a human.

As used herein, the term “introducing” means providing or administeringto an individual. Methods of introducing nanotubes into individuals arewell known to those of ordinary skill in the art and include, but arenot limited to, injection, intravenous or parenteral administration.Single, multiple, continuous or intermittent administration can beeffected.

As used herein, the term “nanotube” includes structures formed fromvarious materials using a generally known template method. Further,nanotubes include structures of various shapes having dimensions smallerthan about 1000 nm, preferably smaller than about 100 nm, morepreferably smaller than about 50 nm, and most preferably smaller thanabout 20 nm. In a preferred embodiment, these nanostructures are hollowtube-like and/or solid wire-like structures.

As used herein, the term “self-rectifying” refers to the ability of thenanotubes to stop acting as a proton channel once the mitochondrialmembrane potential is lowered to a set threshold.

Nanotubes

The present invention relates to nanotubes used as mitochondrialuncouplers in the body. Previous mitochondrial uncouplers, such as2,4-DNP, are often toxic as they decrease and/or stop the production ofATP in the body. The nanotubes of the present invention act are safe, asthey do not adversely affect production of ATP and utilize a differentuncoupling mechanism than the mechanism of the chemical uncouplers.

The presently disclosed nanotubes act as self-rectifying proton channelsacross the mitochondrial inner membrane. When the membrane potentialreaches a certain point, the nanotubes shut off the proton flow. Thismechanism is similar to that of an electrical circuit, as the flowacross mitochondrial membranes follows the principles of Ohm's law.According to Ohm's law, V (voltage)=I (current) R (resistance) (i.e., ifone reduces R, I will increase). In the case of mitochondria, thevoltage is the separation of protons across the inner membrane due tomitochondrial respiration (i.e., electron transport system activity) andthe current is the flow of protons back across the inner membrane backinto the matrix (i.e., flow of protons through the ATP synthase ornanotubes).

Under normal conditions, resistance (“R”) is set by the demand for ATPto be produced by mitochondria. In the case of uncontrolled chemicaluncoupling, resistance is a function of the amount of uncoupler that ispresent. Thus, if the dosage is too high, all resistance is abolishedresulting in maximum current flow. In the case of the presentlydisclosed nanotubes, resistance can be adjusted by changing the innerdiameter of the nanotube and/or by “doping” the inside of the nanotubeswith compounds of specific pKas. The present nanotubes act as protonchannels which allow protons to flow back into the matrix across theinner mitochondrial membrane due to the voltage (i.e., separation ofcharge) that is present.

The flow of protons can be controlled by increasing or decreasing theinner diameter the nanotubes such that they will only allow a flow ofprotons only when a threshold voltage is reached. By decreasing theinner diameter of the nanotubes, the amount of voltage required to“push” protons through the nanotube is increased, such that only whenthe required voltage (or higher) is applied will protons flow throughthe nanotubes back into the matrix. This in effect makes the nanotubesself-rectifying in that they will not act as proton channels once themitochondrial membrane potential is lowered to a set threshold. Thisfailsafe mechanism will activate regardless how many tubes are present,because they will only act as proton channels when the threshold voltageis present.

Thus, the present nanotubes are safe to administer as mitochondrialuncouplers, as they cause the mitochondrial uncoupling to stop prior toany adverse effects.

The nanotubes, acting as proton channels, cause a reduction inmitochondrial membrane potentials, which in turn increases basalmetabolism, decreases reactive oxygen species (“ROS”), and decreasesdetrimental loading of Ca²⁺ into mitochondria.

The present nanotubes may be made of metals, polymers, semiconductors,carbons, and other materials. Preferably, the nanotubes are made of anyelemental metal that will plate or coat the walls of the template used,including, but not limited to, gold, copper, platinum, nickel andsilver. Most preferably, the nanotubes are made of gold, as gold isinert and does not cause any inflammatory response in the body.

The nanotubes can be made from polymers. These polymers can be naturalor synthetic and can include poly(vinyl alcohol), poly(esters),polyglycolide, polycaprolactone, poly(ethylene oxide), poly(butyleneterephthalate), poly(hydroxyalkanoates), hydrogels, modifiedpoly(saccharide)s such as starch, cellulose, and chitosan.

Different characteristics of the nanotubes, such as the effectivehalf-life (i.e., how long the nanotube will be stable in the body), canbe altered by changing the percent composition of the different polymersto achieve a desired effect. See Martin C.R., et al., Materials science:Expanding the Molecular Electronics Toolbox, Science, 2005 Jul. 1,309(5731):67-8; Kohli, P., et al., Smart Nanotubes for Biotechnology,Current Pharmaceutical Biotechnology, 2005 Feb., 6(1):35-47; MartinC.R., Nanomaterials: A Membrane-Based Synthetic Approach, Science(1994), 266, 1961-1966; and Martin C.R., The Emerging Field of NanotubeBiotechnology, Nature Reviews Drug Discoveries (2003), 2 29-37.

Copolymers of poly(ester)s based on polylactide, polyglycolide, andpolycaprolactone can also be used to make the nanotubes. Multiblockcopolymers of poly(ethylene oxide) and poly(butylene terephthalate) canalso be used to make the nanotubes. The poly(esters) can be based onpolylactide.

The use of nanotubes as proton channels in mitochondria can circumventthe toxic side effects of chemical uncouplers and safely increasemetabolism (and promote weight loss). Nanotubes act as high-conductanceproton channels, mimicking chemical uncouplers, without substantiallyany chance of toxicity. This can be accomplished by the use andmanufacturing of voltage-dependent nanotubes that act as effectiveproton diodes and rectifiers in the mitochondrial membrane. Thenanotubes can conduct protons only when a specific potential (i.e.,breakover voltage) is reached which causes the nanotubes to act asproton channels which will reduce mitochondrial membrane potential to apoint that will close the nanotube proton channel.

As discussed supra, one reason that the present nanotubes are safe foruse in the body is the mechanism of conducting protons only when aspecific potential (i.e., breakover voltage) is reached. Thischaracteristic of the nanotubes causes a drop in the mitochondrialmembrane potential that closes the nanotube proton channel, in effectmaking the nanotubes self-rectifying. Therefore, nanotubes can bedesigned to maintain a specific mitochondrial membrane potential thatcan significantly increase metabolism, substantially without thepossibility of toxicity, because the nanotubes can be designed to notfunction at a mitochondrial membrane potential below the threshold forATP production. Additionally, the nanotubes (i.e., proton channels) canonly function in the mitochondria, because a proton gradient of about120-220 mV does not exist anywhere else in mammalian cells.

Nanotubes can be designed to maintain a specific mitochondrial membranepotential that can significantly increase metabolism without thetoxicity associated with reducing mitochondrial membrane potential belowthe threshold for ATP production. Nanotube proton channels can functiononly at the mitochondria in the cells of in an individual because aproton gradient of similar magnitude does not exist anywhere else in anindividual's cell.

The inner diameter (“i.d.”) of the nanotubes can be altered until thedesired conductance is realized. It is well known that reducing thenanotube i.d. also reduces the conductance (increases the resistance) ofthe channel to proton flow in accordance with Ohms law. See NishizawaM., et al., Metal Nanotubule Membranes with Electrochemically SwitchableIon-Transport Selectivity, Science (1995), 268, 700-702; Miller S.A., etal., Electroosmotic flow in template-prepared carbon nanotube membranes,J. Am. Chem. Soc (2001). 123, 12335-12342; and Martin, C.R.,Nanomaterials: A Membrane-Based Synthetic Approach, Science (1994), 266,1961-1966.

Further, the proton conductance and selectivity of the nanotubes can beadjusted and/or increased by using compounds of specific pKa includingspecific chemisorbed amino acids. These amino acids can be incorporatedinto the inner walls of the nanotubes during template synthesis.Depending on the chemical properties of the amino acids, nanotubes thatare highly-selective for protons in addition to being pH sensitive canbe created.

Generally, a pH difference of about 0.5 exists across the innermitochondrial membrane. When the interior surface of the nanotubes iscoated (i.e., “doped”) with compounds of specific pKa such that thenanotube shuts off when this pH gradient reaches a certain level suchthat the compound will no longer be protonated and cease to conductprotons. This acts as a second failsafe device, in addition tocontrolling proton conductance. Based on the pH gradient across themitochondrial inner membrane, any compound with a pKa in the range of 4to 5 (in H₂O at ˜37° C.) and that has a small enough molecular weightthat it can fit into the nanotube, would be ideal for this application.Ion selectivity can also be adjusted by doping the inside of thenanotubes with compounds of the opposite charge of the ion selected.

Preparation of Nanotubes

A template method of forming the nanotubes generally includes (a)immersing a template membrane into methanol, (b) immersing the templatemembrane into a solution having SnCl₂ and trifluoroacetic acid, (c)immersing the template membrane in methanol twice, (d) immersing thetemplate membrane in an aqueous ammonical AgNO₃ solution or any aqueoussolution containing the material you wish to use for plating of themembrane, (e) immersing the template membrane in methanol, (f) placingthe template membrane in a gold-plating bath, the gold-plating bathhaving commercial gold plating solution, (g) adjusting the pH of thegold-plating bath to about 10 by drop-wise addition of H₂SO₄ whilestirring, (h) placing the template membrane in the gold-plating bath fordifferent periods of time to obtain hollow tube-like structures ofdifferent inside diameters. CH₂Cl₂ is added to dissolve the membrane.

In one embodiment, the template method of forming the nanotubes includes(a) immersing a template membrane into methanol for about 2-10 minutes,(b) immersing the template membrane into a solution having SnCl₂ andtrifluoroacetic acid for about 30-60 minutes, (c) immersing the templatemembrane in methanol twice for about 1-5 minutes each time, (d)immersing the template membrane in an aqueous ammonical AgNO₃ solutionfor about 2-10 minutes, (e) immersing the template membrane in methanolfor about 2-10 minutes, (f) placing the template membrane in agold-plating bath at a temperature of about 2-10° C., the gold-platingbath having commercial gold plating solution, which typically includesNa₂SO₃, formaldehyde, and NaHCO₃, (g) adjusting the pH of thegold-plating bath to about 10 by drop-wise addition of H₂SO₄ whilestirring, (h) placing the template membranes in the gold-plating bathfor different periods of time to obtain hollow tube-like structures ofdifferent inside diameters. CH₂Cl₂ is added to dissolve the membrane.

Preferably, in another embodiment, the template method of forming thenanotubes includes (a) immersing a template membrane into methanol forabout 2-10 minutes, preferably for about 5 minutes, (b) immersing thetemplate membrane into a solution having about 0.025 M SnCl₂ and 0.07 Mtrifluoroacetic acid for about 30-60 minutes, preferably for about 45minutes, (c) immersing the template membrane in methanol twice for about1-5 minutes each time, preferably for about 2.5 minutes each time, (d)immersing the template membrane in a 0.029 M aqueous ammonical AgNO₃solution for about 2-10 minutes, preferably for about 5 minutes, (e)immersing the template membrane in methanol for about 2-10 minutes,preferably for about 5 minutes, (f) placing the template membrane in agold-plating bath at a temperature of about 2-10° C., preferably at atemperature of 5° C., the gold-plating bath having commercial goldplating solution, which typically includes 0.127 M Na₂SO₃, 0.625 Mformaldehyde, and 0.025 M NaHCO₃, (g) adjusting the pH of thegold-plating bath to about 10 by drop-wise addition of 0.5 M H₂SO₄ whilestirring, (h) placing the template membranes in the gold-plating bathfor different periods of time to obtain hollow tube-like structures ofdifferent inside diameters. CH₂Cl₂ is added to dissolve the membrane.

In another embodiment, a polycarbonate track etched membrane (e.g., fromSterlitech™ Corporation) is immersed in methanol for 5 minutes.Substantially all of the methanol is drained off and the membrane isimmersed in a solution which is about 0.025 M SnCl₂ and about 0.07 Mtrifluoroacetic acid. Both the chemicals should be added in equalvolumes. The membrane is kept in the SnCl₂ and trifluoroacetic acidsolution for about 30-60 minutes. The solution is then drained off andthe membrane is immersed in methanol. The immersion of the membrane inmethanol is to remove any residual SnCl₂ or trifluoroacetic acid. Themembrane is then immersed in an aqueous ammonical AgNO₃ solution. Themembrane is placed in a solution containing substantially equal volumesof Na₂SO₃, NaHCO₃, HCOOH, and a commercial gold plating solution. Thesolution should be maintained at a temperature of about 5° C. Themembranes are kept in the gold-plating solution for 3, 6, 9 or 24 hours.The inner diameter of the tubes changes with the plating time. After therespective amount of time, the solution is drained-off and CH₂Cl₂ orother similar solvent is added to dissolve the membrane. The solution isthen centrifuged to separate aggregated nanotubes. The solution is thenremoved leaving the aggregated nanotubes behind. PEG is added to theaggregated nanotubes such that the PEG coats the nanotubes causing theaggregated nanotubes to separate such that substantially individualnanotubes coated with PEG are available in water or similar solvent as asolution. The mixture is then vortexed (i.e., mixed vigorously using agenie vortexer or other similar mixer) to aid the coating of thenanotubes with PEG thereby increasing the availability of the nanotubesin solution. The mixture is then filtered to remove any particulatematter. The filtered nanotubes are then pelleted by centrifugation, thesupernatant removed and the pellet re-suspended in ethanol to removeexcess PEG. The nanotubes are then pelleted again by centrifugation, thesupernatant removed and the pellets are re-suspended in sterile waterand stored at about 4° C.

The plated membranes obtained from the method described above can thenbe placed in a solution of histidine (or any compound one wishes to usto dope the inside of the nanotubes). The plated membranes are left inthe histidine solution for 24 hours to allow the histidine to coat theinner walls of the plated membranes having the nanotubes. The coatedmembranes can be removed and placed in CH₂Cl₂ and sonicated, so as todissolve the membrane. PEG can be added to the resultant solution whichwill coat the outside of the nanotubes making them soluble. The PEGsolution can be centrifuged so that the nanotubes settle at the bottom.The PEG solution can be removed so that the nanotubes can be collected.Ethanol (“EtOH”) can be added allowing the tubes to be suspended in asterile medium. The solution can be drained off after centrifuging andEtOH can be added to obtain histidine coated nanotubes.

PEG is added to the outside of the nanotubes in order to allow them tobecome soluble in solution, prior to filtration. Nanotubes that aremanufactured in the absence of PEG do not make it through the filtrationprocess.

Further, the proton conductance of the nanotubes can be adjusted byusing compounds of specific pKa including specific chemisorbed aminoacids. These amino acids can be incorporated into the inner walls of thenanotubes during template synthesis. Depending on the chemicalproperties of the amino acids nanotubes that are highly-selective forprotons in addition to being pH sensitive can be created. Generally, apH difference of about 0.5 exists across the inner mitochondrialmembrane. When the interior surface of the nanotubes is doped withcompounds of specific pKa, that the nanotubes shut off when a specificpH is reached.

Any weak acid that has a pKa of 4-5 in H₂O at about 37° C. and hasmolecules that can penetrate the i.d. of the nanotubes, can be used asproton conductance adjusting compounds. In a preferred embodiment, theseproton conductance adjusting compounds are selected from the groupconsisting of asparate, glutamate, and combinations thereof.

Any organic solvent that can dissolve the template used in the templatemethod of forming nanotubes can be used. In a preferred embodiment, theorganic solvent may be methanol, methylene chloride, or combinationsthereof.

Uses of Nanotubes as Mitochondrial Uncouplers

The present nanotubes may be used to treat or manage any condition thatis related to, or may be affected by, mitochondrial uncoupling.Moreover, any condition caused by an increase in ROS and/or an increasein Ca²⁺ can be treated and/or the effects therefrom can be reduced, byadministering the nanotubes of the present invention.

The following diseases or conditions are exemplary and are not meant tolimit the conditions caused by or aggravated by mitochondrialuncoupling.

Obesity/Weight Control

According National Institute of Health (“NIH”), recent figures from theCenters for Disease Control and Prevention show that 65 percent of U.S.adults—or about 129.6 million people—are either overweight or obese. Inaddition to decreasing quality of life and increasing the risk ofpremature death, obesity and overweight individuals cost the UnitedStates of America (“U.S.”) an estimated $117 billion in direct medicalcosts and indirect costs, such as lost wages due to illness.

Obesity is in epidemic proportions and is recognized as one of the mostimportant health issues facing the U.S. Numerous research studies havedirectly shown that obesity increases the risk of developing a number ofhealth conditions, including type 2 diabetes, hypertension, coronaryheart disease, ischemic stroke, colon cancer, post-menopausal breastcancer, endometrial cancer, gall bladder-disease, osteoarthritis, andobstructive sleep apnea. In fact, obesity-related disease is now onlysecond to smoking as the cause of premature death in the U.S. (Centersfor Disease Control).

In the 1930s it was recognized that increasing the body's basalmetabolism using mitochondrial uncouplers directly resulted in steadyand rapid weight loss. Chemical mitochondrial uncouplers were found tosignificantly increase weight loss in a dose-dependent manner byreducing membrane potential and increasing respiration in mitochondria(i.e., increasing basal metabolism). See Harper, J. A., et al., (2001),Mitochondrial uncoupling as a target for drug development for thetreatment of obesity, Obesity Reviews 2 (4), 255-265; and Kurt, T.L., etal., Dinitrophenol in weight loss: the poison center and public healthsafety, Vet Hum Toxicol 28, 574-5 (1986). This mechanism effectivelyuncouples mitochondrial adenosine triphosphate (“ATP”) production fromelectron transport (i.e., mitochondrial respiration), which results infoodstuffs being turned into heat instead of being used as an energysource or being stored as fat. In effect, chemical uncouplers increasebasal metabolism which in turn results in weight loss (See FIG. 1).

The chemical mitochondrial uncoupler 2,4-dinitrophenol (“2,4-DNP”) wassold over the counter around the 1930s as a weight-loss supplement. FIG.1 shows two line graphs indicating that 2,4-DNP increases metabolism andweight loss in a dose-dependent manner in humans. In the right panel ofFIG. 1 the linear increase in metabolism as a function of 2,4-DNP dosageis demonstrated. The left panel of FIG. 1 demonstrates that as the doseof 2,4-DNP is increased, which increases metabolism, results in a verylinear increase in weigh loss regardless of diet or changes inlifestyle. See Tainter M. L., et al., (1935), Dinitrophenol in thetreatment of obesity: final report, J. Am. Med. Assoc 105, 332-337.

However, 2,4-DNP was pulled from the marked by the Food and DrugAdministration (“FDA”) as people were routinely overdosing on thecompound in an effort to increase the rate of their weight loss(reviewed in Kurt, T.L., et al., Dinitrophenol in weight loss: thepoison center and public health safety, Vet Hum Toxicol 28, 574-5(1986)).

The toxicity of 2,4-DNP stems from the uncoupling mechanism utilized.Chemical uncouplers, such as 2,4-DNP, are toxic due to the mechanism bywhich chemical uncouplers function. These chemical uncouplers are oftenare weak acids that become protonated (i.e., take up a proton) due totheir pKa in the inner membrane space of the mitochondria, which is moreacidic than the matrix. Protonated chemical uncouplers cross the innermembrane where they release the proton back into the more basic matrixthen they cross back into the inner membrane space and the cyclecontinues until the pH gradient and membrane potential is completelydissipated.

Overdosing with chemical uncouplers was prevalent because complete orexcessive uncoupling of mitochondria (i.e., dropping the mitochondrialmembrane potential below about 100 mV) causes an inability to producecellular ATP, which eventually leads to death. See Sullivan, P.G., etal., (2004), Mitochondrial Uncoupling as a Therapeutic Target FollowingNeuronal Injury, Journal of Bioenergetics and Biomembranes, 36(4),353-356; and Mattiasson, G., et al., (2006), The Emerging Roles of UCP2in Health and Disease, Antioxidants and Redox Signaling, 8(1-2), 1-38.

Nanotubes can offer a safe and effective treatment for obesity andweight management by safely increasing basal metabolism. The presentnanotubes conduct protons only when a specific potential is reached.Nanotubes can be designed to maintain a specific mitochondrial membranepotential that can significantly increase metabolism without thepossibility of toxicity. In contrast to the use of nanotubes asuncouplers, chemical uncouplers significantly increase weight loss in adose-dependent manner by reducing membrane potential and increasingrespiration in mitochondria. This uncouples ATP production from electrontransport which results in caloric intake being turned into heat and notan energy source.

The nanotubes can be manufactured to increase metabolism and/or weightloss by altering the range of potentials that open and close thechannel, decreasing the holding potential in obese patients to maximizemetabolism and weight loss.

The nanotubes may be modified to create a desired effect. Designernanotubes can be manufactured to increase or decrease metabolismaccordingly by altering the range of potentials that open or close theproton channels. For example, by decreasing the holding potential inobese patients, metabolism and weight loss can be maximized.

CNS Disorders/TBI

Generally, chemical uncouplers such as 2,4-DNP and carbonyl cyanide4-trifluoromethoxy phenylhydrazone (“FCCP”) are neuroprotective,following central nervous system (“CNS”) injuries such as traumaticbrain injury, SCI, stroke, Parkinson's disease, etc. Without wishing tobe bound by theory, the mechanism of action most likely involves areduction in mitochondrial Ca²⁺ loading and ROS production followingsuch an injury. Both are linked to the mitochondrial membrane potentialsuch that a high membrane potential increases mitochondrial Ca²⁺ uptakeand ROS production. For example, by increasing the mitochondrialmembrane potential by about 30 Mv, the uptake of Ca²⁺ is increased aboutten fold while also maximizing ROS production due to decreased electrontransport (stalled) which increases the slippage of electrons tomolecular oxygen. In contrast, by decreasing the mitochondrial membranepotential the Ca²⁺ loading is reduced along with reduced production ofROS. The toxicity of chemical uncouplers limits their potential fortherapeutic use in individuals, which is compounded by the altered anddynamic changes in drug metabolism that occurs following CNS injuries.

Nanotubes can offer the benefits of uncoupling and reducing membranepotential following CNS injuries, without the toxicity associated withchemical uncouplers. Furthermore, any alteration in metabolism followingCNS injuries which would affect the ability of the body to metabolizechemical agents would not alter the efficacy of nanotubes.

Nanotubes can offer effective therapy for several neurological disordersin which mitochondria have been demonstrated to play a pivotal roleincluding, but not limited to, traumatic brain injury, SCI, stroke,Alzheimer's disease, and Huntington's disease.

Traumatic brain injury (“TBI”) is a serious health care problem in theUnited States with more than 400,000 individuals hospitalized each yearand an estimated cost of greater than 25 billion dollars. There is anenormous focus on the development and discovery of neuroprotectiveand/or pro-regenerative agents, which may have clinical relevancefollowing TBI.

Neuronal degeneration following TBI is believed to evolve in a biphasicmanner consisting of the primary mechanical insult and a progressivesecondary necrosis. It is believed that alterations in excitatory aminoacids (“EAA”), increased oxidative stress, and the disruption of Ca²⁺homeostasis are major factors contributing to the ensuingneuropathology. See Hall, E.D., et al., Preserving Function in AcuteNervous System Injury. In: From Neuroscience to Neurology: Neuroscience,Molecular Medicine, and the Therapeutic Translation of Neurology,(S.Waxman, Ed.), Elsevier/Academic Press, Amsterdam, pp. 35-59, 2004;Sullivan, P.G., et al., (2004), Mitochondrial Uncoupling as aTherapeutic Target Following Neuronal Injury, Journal of Bioenergeticsand Biomembranes, 36(4), 353-356; Lipshitz, J., et al., (2005),Mitochondrial Damage and Dysfunction in Traumatic Brain Injury,Mitochondrion, 4, 705-713; Mattiasson, G., et al., (2005), The EmergingRoles of UCP2 in Health and Disease, Antioxidants and Redox Signaling,8, 1-38.

Mitochondria play a key role in the cell death cascade, andmitochondrial dysfunction has been directly linked to EAA-mediatedneurotoxicity. This dysfunction is directly related to Ca²⁺ ions thatalter mitochondrial function and increase ROS production. Following TBI,there is a significant loss of mitochondrial homeostasis, resulting inincreased mitochondrial ROS production and disruption of synaptichomeostasis, implicating a pivotal role for mitochondria in the sequelaeof TBI-related neuropathology.

It has been demonstrated in the past that mitochondrial dysfunction is apivotal link in the neuropathological sequelae of brain injury. SeeSingh, I.N., et al., (2006), Time Course of Post-Traumatic MitochondrialOxidative Damage and Dysfunction in a Mouse Model of Focal TraumaticBrain Injury: Implications For Neuroprotective Therapy, Journal ofCerebral Blood Flow & Metabolism, (In Press, Epub Mar 15, 2006); Hall,E.D., et al., Preserving Function in Acute Nervous System Injury. In:From Neuroscience to Neurology: Neuroscience, Molecular Medicine, andthe Therapeutic Translation of Neurology, (S.Waxman, Ed.),Elsevier/Academic Press, Amsterdam, pp. 35-59, 2004; Sullivan, P.G., etal., (2004), Mitochondrial Uncoupling as a Therapeutic Target FollowingNeuronal Injury, Journal of Bioenergetics and Biomembranes, 36(4),353-356; Lipshitz, J., et al., (2005), Mitochondrial Damage andDysfunction in Traumatic Brain Injury, Mitochondrion, 4, 705-713;Mattiasson, G., et al., (2005), The Emerging Roles of UCP2 in Health andDisease, Antioxidants and Redox Signaling, 8, 1-38.

TBI-induced glutamate release increases mitochondrial Ca²⁺cycling/overload ultimately leading to mitochondrial dysfunction. Lossof mitochondrial homeostasis, increased mitochondrial ROS production, aswell as disruption of synaptic homeostasis, occur following TBI.

Extrinsic mitochondrial uncouplers are compounds that facilitate themovement of protons from the mitochondrial inner-membrane space into themitochondrial matrix. Intrinsic uncoupling can be mediated via theactivation of endogenous mitochondrial uncoupling proteins (“UCP”) whichutilize free fatty acids to translocate protons. This short circuit“uncouples” the pumping of protons out of the matrix via the electrontransport system (“ETS”) from the flow of protons through the ATPsynthase and results in a coincidental reduction in the mitochondrialmembrane potential. Long-term complete uncoupling of mitochondria wouldbe detrimental, since it result in a loss in the ability to maintain ATPlevels by mitochondria, whereas a transient or “mild uncoupling”, couldconfer neuroprotection. Mild uncoupling during the acute phases ofTBI-induced excitotoxicity would reduce mitochondrial Ca²⁺ uptake(cycling) and ROS production, as both are Δψ-dependent. Consistent withthese ideas, rats administered a mitochondrial uncoupler post-injury (5min) have less tissue loss, improved behavioral outcomes and demonstratea reduction in mitochondrial oxidative damage, Ca²⁺ loading anddysfunction following TBI (See FIG. 2).

Although the mechanisms contributing to ischemic neuronal degenerationare myriad, mitochondrial dysfunction is now recognized as a pivotalevent that can lead to either necrotic or apoptotic neuronal death. SeeKorde, A.S., The mitochondrial uncoupler 2,4-dinitrophenol attenuatestissue damage and improves mitochondrial homeostasis following transientfocal cerebral ischemia, (2005) J Neurochem, 94(6):1676-84. Further, ithas been shown that 2,4-DNP reduces infarct volume approximately 40% ina model of focal ischemia-reperfusion injury in the rat brain. See id.

However, as discussed above, chemical uncouplers such as 2,4-DNP canuncouple uncontrollably to the point of causing death. Therefore,nanotubes can be used to achieve the same beneficial effects after TBIrelated injuries such as have less tissue loss, improved behavioraloutcomes and demonstrate a reduction in mitochondrial oxidative damage,Ca²⁺ loading and dysfunction following TBI, without any danger oftoxicity.

Cancer

Nanotubes can also be highly effective for the treatment of variouscancers. Present treatment strategies utilize drugs or radiation thatare toxic to replicating cells in the hope that the cancer cells can bekept in check. Such untargeted treatments are not highly effective andcontribute to significant side effects due to damage to otherproliferating non-cancerous cells.

Recent technology enables targeting and labeling of cancerous cells veryspecifically in vivo using nanosphere technology. See Gao, X., et al.,In vivo targeting and imaging with semiconductor quantum dots, (2004)Nature Biotechnology, 22(8), 969-976; Han, M., et al.,Quantum-dot-tagged microbeads for multiplexed optical coding ofbiomolecules, (2001) Nature Biotechnology, 19, 631-635; and Savic, R.,et al., Micellar Nanocotainers distribute to defined cytoplamsicorganelles, (2003) Science, 300, 615-618. With such targeting andlabeling of cancerous cells possible, nanotubes can be specificallydelivered directly to such targeted and labeled cancerous cells.Nanotubes may be designed to reduce membrane potential to a level thatwould result in the death of the targeted and labeled cancerous cellsfrom the inside out. Nanotubes that can act as non-specific channelsthat when opened by the mitochondrial membrane potential, can result inthe rapid swelling and bursting of the mitochondria can also bedesigned. This would release pro-apoptotic proteins from themitochondria and kill the cancerous cell from the inside.

Organism Life Span

Endogenous uncoupling proteins (UCP) have recently been shown toincrease the life-span of flies. It has also been demonstrated thatchemical uncouplers as well as endogenous activation of UCP areneuroprotective in seizure models and may play a role in seizurereducing epilepogenesis. See Sullivan, P.G., et al., Mitochondrialuncoupling protein-2 protects the immature brain from excitotoxicneuronal death, (2003) Annals of Neurology, 53, 711-717; Sullivan, P.G.,et al., The Ketogenic Diet Enhances Increases Mitochondrial UncouplingProtein Levels And Activity In Mouse Hippocampus, (2004) Annals ofNeurology, 55, 576-580; Brown, M.B., et al., Brain region-specific,age-related, alterations in mitochondrial responses to elevated calcium,(2004) Journal of Bioenergetics and Biomembranes, 36, 401-406; Jin, Y.,et al., The Mitochondrial Uncoupling Agent 2,4-Dinitrophenol ImprovesMitochondrial Function, Attenuates Oxidative Damage, and Increases WhiteMatter Sparing in the Contused Spinal Cord, (2004) Journal ofNeurotrauma, 21, 1396-1404; and Korde, A.S., et al., The uncouplingagent 2,4-dinitrophenol improves mitochondrial homeostasis followingstriatal quinolinic acid injections, (2005) Journal of Neurotrauma, 22,1142-1149.

Administration of Nanotubes

The nanotubes can be administered to an individual in a variety of wayswell known in the art and is not limited to any particular technique.

In one embodiment, the nanotubes can be administered to an individual bywrapping the nanotubes in lipid microspheres/tubes. Once the nanotubesare surrounded by the lipid spheres, they become lipid soluble and canbe injected (i.e., intravenous delivery) or ingested for administrationto an individual. The nanotubes can be active only where a protongradient of >140 mV is present (i.e., at the mitochondrial membrane).Therefore, the delivery of the nanotubes does not have to be targetedspecifically to the mitochondria.

In another embodiment, the nanotubes can be administered to anindividual by attaching the nanotubes to viral proteins for delivery.For example, attachment of the nanotube to the trans-activator of thetranscription (“Tat”) peptide not only allows for entry into cells, butalso specifically targets the nanotubes to the mitochondria due to thepositive charge on the Tat peptide.

In another embodiment, the nanotubes, e.g., the gold nanotubes, can beadministered to the individual via particle bombardment. This techniqueis very simple and has been used for vaccinations. See Lin, M.T., etal., The gene gun: current applications in cutaneous gene therapy,(2000) Int. J. Dermatol., 39(3):161-70. Particle bombardment uses a“gene gun” to deliver the gold nanotubes using a shockwave. This allowsfor substantially precise placement of the nanotubes into various layersof the skin or muscle depending on the pressure used to generateshockwave.

In another embodiment, the nanotubes can be administered to theindividual by patch or gel application to the dermis. Given the size ofnanotubes this is a simple approach to delivery of the nanotubes over aspecific time-period. The patch or gel application to the dermis alsoallows for specific dosage and delivery of the nanotubes by varying therelease of the nanotubes from the patches or gel into the dermis (e.g.,a nicotine patch).

In another embodiment, the outside surface of the nanotubes is coatedwith polyethylglycol (“PEG”). The PEG coated nanotubes can be easilyadministered to the individual regardless of the route of administrationsince PEG coating of the nanotubes makes them water soluble and able toreadily cross the blood brain barrier in mammals. This coupled with thefact that the dosage needed to cause an effect is small should allowefficient uptake via oral administration (e.g., capsules, tablets,suspension of nanotubes).

A therapeutically effective amount or dosage is administered to theanimal. Based on preliminary data in rodents this therapeuticallyeffective dosage is in the range of at least about 0.1 mg/Kg, preferablyabout 1 to 100,000 mg/kg, more preferably about 2 to 10,000 mg/Kg, andmost preferably about 2.5 to 5,000 mg/Kg. As the nanotubes areself-rectifying, once the lowest effective dosage is reached (i.e., thedosage that causes the desired or targeted increase in metabolism)higher dosages will have no further effect.

The nanotubes are present in the compositions and formulations in anamount sufficient to act as mitochondrial uncouplers and/or treat,manage and/or prevent a disease condition. The nanotubes are effectiveover a wide dosage range and are generally administered in apharmaceutically or therapeutically effective amount. The therapeuticdosage of the nanotubes will vary according to, for example, theparticular use for which the treatment is made, the manner ofadministration of the nanotubes, the health and condition of thepatient, and the judgment of the prescribing physician. For intravenousadministration, the dose will typically be in the range of about1.0-10.0 mg/kg. Due to an inevitable decrease in absorbance of thedosage of nanotubes from a gastrointestinal tract, the dosage would haveto be increased about 5 to 10 fold when the nanotubes are administeredin any oral form. Effective doses can be readily extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The actual amount of the nanotubes administered will depend on a numberof factors, such as the severity of the disease, the age and relativehealth of the subject, and the route and form of administration, andother factors.

The amount administered to the patient will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis versus therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, nanotubes areadministered to a patient already suffering from symptoms and/or acondition in an amount sufficient to cure or at least partially arrestthe symptoms and complications. An amount adequate to accomplish this isdefined as “therapeutically effective dose.” Amounts effective for thisuse will depend on the age, weight and general condition of thesubject/patient, and the like.

These nanotubes may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. When employed aspharmaceuticals, the nanotubes of the subject invention are usuallyadministered in the form of pharmaceutical compositions. This inventionalso includes pharmaceutical compositions comprising nanotubes,associated with one or more pharmaceutically acceptable carriers orexcipients. The excipient is typically one suitable for administrationto human subjects or other mammals. In making the compositions of thisinvention, the active ingredient is usually mixed with an excipient,and/or diluted by an excipient. When the excipient serves as a diluent,it can be a solid, semi-solid, or liquid material, which acts as avehicle, carrier or medium for the active ingredient.

The nanotubes of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

Suitable methods and formulations for use in the present invention arefound in REMINGTON'S PHARMACEUTICAL SCIENCES, Mace Publishing Company,Philadelphia, PA, 17th ed. (1985).

According to one aspect of the invention, the nanotubes may beadministered alone, or in combination with any other medicament. Thus,the formulation may comprise nanotubes in combination with anotheractive ingredient, such as a drug, in the same formulation. Whenadministered in combination, the nanotubes may be administered in thesame formulation as other compounds as shown, or in a separateformulation. When administered in combination, the nanotubes may beadministered prior to, following, or concurrently with the othercompounds and/or compositions.

EXAMPLES Example 1

Preparation of Nanotubes by Template Method

To prepare nanotubes using the template method, polycarbonate tracketched membrane were immerse in methanol for 5 minutes. All of themethanol was drained off and then the membrane was immersed in asolution comprising 0.025 M SnCl₂ and 0.07 M trifluoroacetic acid. Bothof these chemicals were added in equal volumes. The membrane was kept inthe SnCl₂ and trifluoroacetic acid solution for 45 minutes. The solutionwas drained off and the membrane was immersed in methanol for twoconsecutive times, each for 2.5 minutes. The immersion of the membranein methanol two consecutive times was done to remove any residual SnCl₂or trifluoroacetic acid. The membrane was then immersed in an aqueousammonical AgNO₃ solution for 5 minutes.

Next, the membrane was placed in a solution containing equal volumes ofthe following: 0.127 M of Na₂SO₃, 0.025 M of NaHCO₃, and 0.625 M ofHCOOH and a commercial gold plating solution Na₃Au(SO₃)₂ (diluted fromOromerse Part B, Technic, Inc.). The solution was maintained at atemperature of about 5° C. The inner diameter of the tubes changed withthe plating time. The membranes were kept in the gold-plating solutionfor 3, 6, 9 or 24 hours. After the respective amount of time, thesolution was drained-off and CH₂Cl₂ was added to dissolve the membrane.The solution was then centrifuged to separate the aggregated nanotubes.

The solution was then carefully removed, leaving the aggregatednanotubes behind. About 2.5 mL of PEG was added to the aggregatednanotubes such that the PEG coated the nanotubes, causing the aggregatednanotubes to separate such that substantially individual nanotubescoated with PEG are available in water or similar solvent as a solution.The mixture was then vortexed (i.e., mixed vigorously using a genievortexer or other similar mixer) to aid the coating of the nanotubeswith PEG, increasing the availability of the nanotubes in solution.

The mixture was filtered using centricons by centrifugation to removeany particulate matter. The filtered nanotubes were then pelleted bycentrifugation, the supernatant removed and the pellet re-suspended in70% ethanol to remove excess PEG. The nanotubes were then pelleted againby centrifugation, the supernatant removed and the pellets arere-suspended in sterile water and stored at 4° C.

0.025 M of SnCl₂ was required. For a preparation of 100 ml, 0.948 gm ofSnCl₂ was used, and for 40 ml, 0.3792 gm of SnCl₂ is used. The aqueousammonical AgNO₃ solution was prepared by adding 0.0984 gm of AgNO₃, thenadding a panel volume of 5 N NaOH drop-wise (if precipitating).

The resulting solution was used immediately. 0.07 M trifluoroacetic acidwas used in an amount of 0.15964 gm or 0.104.33 μL. 0.127 M Na₂SO₃ wasused in an amount of 0.32014 gm. 0.025 M NaHCO₃ was used in an amount of0.04205 gm. 0.625 M formaldehyde was used in an amount of 0.3752 gm,wherein the total volume is 173.7 mL.

CH₂Cl₂ was added to dissolve the membrane. Sufficient CH₂Cl₂ (about 10mL) was added to cover the membrane adequately and the resulting mixtureis centrifuged and/or sonicated. Then, the nanotubes obtained from themembrane were again centrifuged and after separating the liquid, about2-5 mL of PEG was added. Attomol can be added instead of PEG.

Before dissolving the membranes with CH₂Cl₂, as described above, thenanotubes were optionally coated with histidine by adding about 10 mL ofa histidine solution such that the membranes are substantially immersedin the histidine. Further, acetic acid is added to balance the pH of thehistidine solution. The membranes remain immersed in thehistidine/acetic acid solution for a time sufficient to substantiallycoat the nanotubes.

Example 2

Preparation of Nanotubes using histidine

The nanotubes were prepared using the method set forth in Example 1.Then the plated membranes were placed in a solution of histidine, andleft in the solution for 24 hours, to allow the histidine to coat theinner walls of the nanotubes plated in the membranes. The coatedmembranes were removed and placed in CH₂Cl₂ and sonicated, to dissolvethe membrane. PEG was added to the resultant solution to release thenanotubes into the PEG solution. The PEG solution was sonicated so thatthe nanotubes settled at the bottom. The PEG was removed so that thenanotubes could be collected. 70V EtOH was added, allowing the tubes tobe suspended in a sterile medium. The solution was drained off aftercentrifuging, and then 70V EtOH was added to it.

Example 3

Preparation of Gold nanotubes using Template Membranes

Materials used in this example included Polycarbonate Track Etchedmembranes (from Sterlitech™ Corporation); SnCl₂, ammonium hydroxide,tri-flouroacetic acid from Sigma Aldrich; L-Histidine, Stannous ChlorideAnhydrous and Silver Nitride from Fluka; Sodium Sulfite

Anhydrous and Sodium Bicarbonate from Mallinckrodt; Ormerse SO Part B™,commercial gold solution.

The procedure for the preparation of the gold nanotubes includedimmersing the polycarbonate track etched membrane in methanol for about5 minutes. Substantially all of the methanol was drained off, and thenthe membrane was immersed in a solution which is 0.025 M in SnCl₂, i.e.,0.1896 gm for 20 mL of water and 0.07 M in trifluoroacetic acid (i.e.,104.33 μL for 20 mL of water. Both the SnCl₂ and trifluoroacetic acidwere added in equal volumetric proportions. The membrane was keptimmersed in the SnCl₂ and trifluoroacetic acid solution for about 45minutes. Then the liquid was drained off and the membrane immersed againin methanol for 2 consecutive times, each time for about 2.5 minutes toclean the membrane from the previously added chemicals. Aqueousammonical AgNO₃ solution was added to the membrane and the membrane wasleft in the solution for about 5 minutes. The membrane was immersed in asolution at a temperature of 5° C. The solution contained 3 mL of eachof the following: (i) 0.127 M of Na2SO₃, i.e., 0.32014 gm for a 20 mLsolution; (ii) 0.025 M of NaHCO₃, i.e., 0.04205 gm for a 20 mL solution;0.625 M of HCOOH, i.e., 347.4 μL for a 20 mL solution; commercial goldplating solution.

The inner diameter of the nanotubes changes with plating time.Therefore, the membranes were kept in the above solution for about 24hours in order to achieve ideal proton conductance rates (i.e., theamount of mitochondrial uncoupling). After the respective amount oftime, the solution was drained and CH₂Cl₂ was added to dissolve themembrane. Then the solution was centrifuged so that the nanotubessettled down at the bottom. PEG was added to the solution and theresultant solution was stored at 4° C. The liquid was then filteredthrough a 0.08 μm filter so that the fine nanotubes without any residue(particulate matter) could be collected.

Example 4

Coating the nanotubes with Histidine

After obtaining the nanotubes prepared in Example 3, histidine was addedas an aqueous solution to the nanotubes and centrifuged at a very lowspeed for about 5 minutes. The solution was vortexed a few times toensure that the histidine coated the nanotubes. Then the procedure ofExample 2 was followed.

Instead of using the 0.08 μm filter in Example 3, larger filters can beused having a size ranging between 0.001 μm and 0.1 μm.

Example 5

In vitro study of interaction of nanotubes with mitochondria

Mitochondria were prepared from adult Sprague-Dawley rats and nanotubesobtained from the above examples (both histidine-coated and uncoatedtubes) where added to examine state 3 and state 4 respiration rates ofthe mitochondria and the effect of the nanotubes were measured.

08/24 1 animal 08/25 1 08/30 1 08/31 1 09/02 1 Mito resp + NT EM PIC

The mitochondria were prepared by Ficoll preparation and then themitochondria were collected for respiration, ethyl ester oftetramethylrhodamine (TMRE) based membrane potential estimation andespecially for electron micrograph pictures.

Samples for EM

-   -   1. Control—mitochondria without nanotubes (ethanol 75%) or for        the EM control included the addition of nanotubes that had been        made in the absence of PEG such that the nanotubes were not        soluble and where filtered out of the solution.    -   2. Mitochondria with nanotubes that were plated for different        times or in the absence or presence of histidine. Following        measurements of mitochondrial respiration the samples were        pelleted by centrifugation and processed for EM.

Example 6

As shown in FIG. 2, mitochondrial uncoupling increases tissue sparingfollowing traumatic brain injury. Adult Sprague-Dawley rats received amoderate injury (about 1.5 mm compression of the cortex) and wereadministered either vehicle (DMSO), 5 mg/kg of 2,4-DNP, 2.5 mg/kg FCCPor 6.2 mg/kg of at 5 min post-injury. Representative sections from aninjured 2,4-DNP-treated animals and vehicle-treated at 15 dayspost-injury are shown in panel A. The mitochondrial uncouplers 2,4-DNPand FCCP significantly increased tissue sparing compared tovehicle-treated animals. In contrast, administration of TNP, anananlogue of 2,4-DNP that does not uncouple mitochondria, had nosignificant effect on tissue sparing. Bars represent group means, SD(n=6/group) and * indicates p<0.01 compared to vehicle treated animals.

Example 7

As shown in FIG. 3, post-injury administration (15 mins post-injury) of5 mg/kg of 2,4-DNP increases tissue sparing following SCI. Bar graphsrepresenting the extent of tissue sparing measured throughcross-sections of the T10 segment injury epicenters 48 hours after amoderate (150 kydn) contusion SCI. The 2,4-DNP-treated groups showedsignificantly greater sparing compared to the vehicle (DMSO) treatedanimals through the lesion epicenters. The significantly greatercross-sectional area of tissue preservation with both treatments (leftgraph) was reflected in a significantly higher percentage of tissuesparing at the lesion epicenters compared to vehicle controls (rightgraph). The lesion epicenter means were derived from sections in eachcord demonstrating the least spared tissue. Bars represent group means,SEM (n=5-6/group). Using an unpaired t-test, * indicates p<0.05 comparedto vehicle-treated groups.

Example 8

FIG. 4 shows electron microscopy photomicrographs of corticalmitochondria isolated from adult Sprague-Dawley rats (na{dot over(i)}ve) following the addition of nanotubes (24 hr+histidine) that weremade in the presence of PEG (right panel) and in the absence of PEG(left panel). PEG is coated on the outside of the nanotubes in order toallow them to become soluble in solution by breaking up the aggregatednanotubes into individually PEG coated nanotubes. The PEG coatednanotubes can be filtered and administered. Nanotubes that aremanufactured in the absence of PEG do not make it through the filtrationprocess. In the right panel, nanotubes are evident as the dense, darkparticles and as indicated by the arrows can be found in both thecristae (i.e., folded inner membrane) and outer membrane of themitochondria. The presence of the intact outer and inner membranes alsoindicates that nanotubes do not alter the ultrastructure of themitochondria.

Example 9

FIG. 5 shows a higher magnification of cortical mitochondria isolatedfrom adult Sprague-Dawley rats (na{dot over (i)}ve) following theaddition of nanotubes (24 hr+histidine) that were made in the presenceof PEG. This image illustrates the location of the nanotubes (dark,dense spots) in the mitochondria and the arrowhead indicates a nanotubelocated in the cristae (inner membrane of mitochondria).

Example 10

The bar graph depicted in FIG. 6 shows that mitochondrial respiration isincreased as a function of nanotube plating time (i.e., the longer theplating time the smaller the i.d. of the nanotube) and/or the “doping”of the inside of the nanotube with compounds of specific pKas.Mitochondrial respiration was measured using standard oxymetrictechniques in cortical mitochondria isolated from adult Sprague-Dawleyrats (na{dot over (i)}ve) to determine if nanotubes would increaserespiration and uncouple mitochondria. As shown in FIG. 4, short platingtimes increased the amount of mitochondrial respiration and oxygenconsumption. All measurements were made in the presence of oligomycin,which is an inhibitor of the mitochondrial ATP synthase, to induce stateIV respiration, which is the state of respiration in which mitochondriautilize the minimal amount of oxygen and protons cannot cross the innermembrane of the mitochondria. The data shown in FIG. 4 is expressed asnmols of oxygen consumed per minute per mg of mitochondrial protein.Bars are group means, SEM, (n=4-5/group).

Example 11

FIG. 7 shows nanotubes (labeled as “NT” in FIG. 5) manufactured byplating for 24 hrs and/or “doped” with histidine do not impairmitochondrial ATP production. All assays were performed during state IIIrespiration which is induced by the addition of ADP to corticalmitochondria isolated from adult Sprague-Dawley rats (na{dot over(i)}ve) and oxygen consumption was measured as the mitochondriaconverted ADP into ATP. The data shown in FIG. 5 is expressed as nmolsof oxygen consumed per minute per mg of mitochondrial protein. As shownin FIG. 5, the nanotubes increased respiration/metabolism withoutreducing mitochondrial ATP production. Bars are group means, SEM,(n=6/group).

Example 12

FIG. 8 shows nanotubes (labeled as “NT” in FIG. 6) manufactured byplating nanotubes for 24 hrs and/or doping them with histidine increaserespirations less than nanotubes manufactured for shorter times.Mitochondria were isolated from adult Sprague-Dawley rats (na{dot over(i)}ve) and mitochondrial oxygen consumption was measured following theaddition of the various nanotubes and compared to maximum oxygenconsumption induced by the addition of the chemical mitochondrialuncoupler FCCP. The data shown in FIG. 6 is expressed as the % ofFCCP-induced maximum oxygen consumption (respiration). Bars are groupmeans, SEM, (n=6/group).

Example 13

FIG. 9 shows that sequential additions of nanotubes results in asaturation point being reached due the self-rectifying nature of thenanotubes. In other words, once a saturation point is reached (i.e.,enough nanotubes have been added to uncouple all the mitochondriapresent in the preparation) no more increase in respiration occurs sincethe nanotubes shut themselves off to proton flow when membrane potentialdrops below threshold (as discussed above). Mitochondrial respiration(oxygen consumption) was measured in cortical mitochondria isolated fromadult Sprague-Dawley rats (na{dot over (i)}ve) that were locked in stateIV respiration using oligomycin. To begin the experiment a 1 μl additionof PEG, which functions as a vehicle or carrier for the nanotubes, wasadded followed by four-1 μl additions of nanotubes every 5 minutes. Thedata shown in FIG. 7 is expressed as the % increase in respirationcompared to state IV respiration. Points are group means, SEM, (n=3individual experiments.

Example 14

FIG. 10 shows that nanotubes reduce mitochondrial membrane potentialwhich explains their ability to increase respiration, oxygenconsumption, and metabolism. Cortical mitochondria isolated from adultSprague-Dawley rats (na{dot over (i)}ve) were used for the experimentssummarized in FIG. 8, which utilized the cationic membrane potentialfluorescent indicator TMRE. TMRE, due to its positive charge, issequenced into the mitochondrial matrix following the addition ofpyruvate and malate (P+M), which results in a reduction in fluorescentsignal. Compared to the control samples (in which an equal volume ofsaline was added to the buffer), nanotubes (plated 24 hr+histidine)reduce the mitochondrial membrane potential evident by the increase inTMRE signal illustrated in all conditions. This is due to less membranepotential being available to drive the uptake of TMRE into themitochondrial matrix treated with the nanotubes.

It should be noted that mitochondria treated with nanotubes respondsubstantially the same way to all other conditions expect for the lowermembrane potential. It should further be noted that an additional bolusof nanotubes did not affect membrane potential showing that thenanotubes are self-rectifying. The steep deflections are artifact causedby the opening of the spectrofluormeter door to make additions to thechamber which allows light from the room to enter the spectrofluormeterchamber.

Example 15

FIG. 11 shows that nanotubes reduce reactive oxygen species productionin isolated mitochondria. Mitochondria are the primary source ofreactive oxygen species in cells as electrons slip from the electrontransfer chain and reduce oxygen to superoxide. Cortical mitochondriawere isolated from adult Sprague-Dawley rats (na{dot over (i)}ve) andreactive oxygen species production measured using the fluorescentindicator DCF, which fluoresces when oxidized leading to an increase insignal. Mitochondrial reactive oxygen species were assessed under bothbasal conditions and in the presence of oligomycin to maximize membranepotential and reactive oxygen species production. Nanotubes reduced bothbasal and maximal reactive oxygen species produced by mitochondria.

Example 16

FIG. 12 shows that animals injected with nanotubes stop gaining weightcompared to vehicle controls. Adult Sprague-Dawley rats wereadministered either saline or 2.7 mg/kg nanotubes intraperitoneallyfollowing weighing on day 0. The animals were then weighed everyday atthe same time for 7 days. Food and water intake did not differ betweenthe groups and no toxic side-effects in either group was noted (e.g.,panting, ruffled fur, vocalization, etc.). All the animals in the groupswere considered to be in good health. On average the saline-treatedanimals increased their body weight by ˜5-6% over the 8 day periodwhereas the nanotube-treated animals showed less than 1% gain in bodyweight over the same period of time.

As shown in FIG. 13, adult Sprague-Dawley rats were injected withnanotubes at day 0 and then again on day 23 to determine the effectivehalf-life of the nanotubes in vivo. Adult Sprague-Dawley rats wereadministered intraperitoneally either saline (i.e., Control) ornanotubes (2.7mg/kg) following weighing on day 0. The animals were thenweighed everyday at the same time for 31 days. Food and water intake didnot differ between the groups and no toxic side-effects in either groupwas noted (e.g., panting, ruffled fur, vocalization, etc.) and all theanimals were considered to be in good health.

Still referring to FIG. 13, 8 days after the first injection thenanotube treated animals began to gain weight at a rate similar to thatmeasured in control animals, indicating that the nanotubes were nolonger uncoupling mitochondria and raising metabolism. 23 days after theinitial injection of nanotubes we administered the same dosage ofnanotubes to the animals or administered saline to the control animals.Again the nanotube-treated animals lost weigh and weight gain was haltedcompared to control animals. Data points are the group means(n=3/group).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

All references cited and/or discussed above are herein incorporated byreference in their entirety.

What is claimed is:
 1. A method of reducing the effects of traumaticbrain injury in an individual comprising: administering atherapeutically effective amount of nanotubes into the individual,wherein the nanotubes are self-rectifying nanotubes, having a length ofless than 50 nm and a conductance such that the nanotubes conductprotons only when a proton gradient of about 120-220 mV is present. 2.The method of claim 1, wherein the individual is a mammal.
 3. The methodof claim 1, wherein the nanotubes have an inner diameter designed foruncoupling mitochondria.
 4. The method of claim 1, wherein the nanotubeshave an inner diameter designed for self-rectifying the nanotubes. 5.The method of claim 4, wherein the nanotubes are made from metals orpolymers.
 6. The method of claim 5, wherein the metals include gold andsilver.
 7. The method of claim 5, wherein the polymers include naturalor synthetic polymers.
 8. The method of claim 5, wherein the polymerscan be selected from the group consisting of poly(vinyl alcohol),poly(esters), polyglycolide, polycaprolactone, poly(ethylene oxide),poly(butylene terephthalate), poly(hydroxyalkanoates), hydrogels,modified poly(saccharides), starch, cellulose, and chitosan.
 9. A methodof reducing the effects spinal cord injury in an individual comprising:administering a therapeutically effective amount of nanotubes into theindividual, wherein the nanotubes are self-rectifying nanotubes, havinga length of less than 50 nm and a conductance such that the nanotubesconduct protons only when a proton gradient of about 120-220 mV ispresent.
 10. The method of claim 9, wherein the individual is a mammal.11. The method of claim 9, wherein the nanotubes have an inner diameterdesigned for uncoupling mitochondria.
 12. The method of claim 9, whereinthe nanotubes have an inner diameter designed for self-rectifying thenanotubes.
 13. The method of claim 12, wherein the nanotubes are madefrom metals or polymers.
 14. The method of claim 13, wherein the metalsinclude gold and silver.
 15. The method of claim 13, wherein thepolymers include natural or synthetic polymers.
 16. The method of claim13, wherein the polymers can be selected from the group consisting ofpoly(vinyl alcohol), poly(esters), polyglycolide, polycaprolactone,poly(ethylene oxide), poly(butylene terephthalate),poly(hydroxyalkanoates), hydrogels, modified poly(saccharides), starch,cellulose, and chitosan.
 17. A method of reducing the effects of strokein an individual comprising: administering a therapeutically effectiveamount of nanotubes into the individual, wherein the nanotubes areself-rectifying nanotubes, having a length of less than 50 nm and aconductance such that the nanotubes conduct protons only when a protongradient of about 120-220 mV is present.
 18. The method of claim 17,wherein the individual is a mammal.
 19. The method of claim 17, whereinthe nanotubes have an inner diameter designed for uncouplingmitochondria.
 20. The method of claim 17, wherein the nanotubes have aninner diameter designed for self-rectifying the nanotubes.
 21. Themethod of claim 20, wherein the nanotubes are made from metals orpolymers.
 22. The method of claim 21, wherein the metals include goldand silver.
 23. The method of claim 21, wherein the polymers includenatural or synthetic polymers.
 24. The method of claim 21, wherein thepolymers can be selected from the group consisting of poly(vinylalcohol), poly(esters), polyglycolide, polycaprolactone, poly(ethyleneoxide), poly(butylene terephthalate), poly(hydroxyalkanoates),hydrogels, modified poly(saccharides), starch, cellulose, and chitosan.